Formation of stable submicron peptide or protein particles by thin film freezing

ABSTRACT

The present invention includes compositions and methods for preparing micron-sized or submicron-sized particles by dissolving a water soluble effective ingredient in one or more solvents; spraying or dripping droplets solvent such that the effective ingredient is exposed to a vapor-liquid interface of less than 50, 100, 150, 200, 250, 200, 400 or 500 cm −1  area/volume to, e.g., increase protein stability; and contacting the droplet with a freezing surface that has a temperature differential of at least 30° C. between the droplet and the surface, wherein the surface freezes the droplet into a thin film with a thickness of less than 500 micrometers and a surface area to volume between 25 to 500 cm −1 .

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of particleformation, and more particularly, to the formation of stable submicronprotein particles.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with methods to produce stable submicron peptide andprotein particles.

For example, the U.S. Pat. No. 6,723,347 teaches a process for producingprotein powder. The '347 patent describes a process for convenientlyproducing a stable protein powder retaining the higher-order structureat a high level which comprises freezing a protein-containing solutionat a cooling rate of about −300 to −10° C./min. and then drying.

Another example can be found in U.S. Pat. No. 6,284,282, in which Maa etal. teach a method of spray freeze drying proteins for pharmaceuticaladministration. Maa's application relates to the spray freeze drypreparation of dry powder formulations of therapeutic proteins suitablefor administration via pulmonary delivery.

Yet another example is found in U.S. Pat. No. 6,862,890 entitled“Process for Production of Nanoparticles and Microparticles by SprayFreezing into Liquid”. The '890 patent provides a system and a methodfor the production of microparticles and nanoparticles of materials thatcan be dissolved. The system and method provide quicker freezing times,which in turn produces a more uniform distribution of particle sizes,smaller particles, particles with increased porosity and a more intimatemixing of the particle components. The system and method of the '890patent also produce particles with greater surface area thanconventional methods, and a method for the preparation of particles. Aneffective ingredient is mixed with water, one or more solvents, or acombination thereof, and the resulting mixture is sprayed through aninsulating nozzle located at or below the level of a cryogenic liquid.The spray generates frozen particles.

Yet another example is shown in the U.S. Pat. No. 6,254,854 by Edwardset al. entitled “Porous particles for deep lung delivery”. The '854patent teaches improved porous particles for drug delivery to thepulmonary system, and methods for their synthesis and administration.The porous particles are made of a biodegradable material and have amass density less than 0.4 g/cm³. The particles may be formed ofbiodegradable materials such as biodegradable polymers. For example, theparticles may be formed of a functionalized polyester graft copolymerconsisting of a linear a hydroxy-acid polyester backbone having at leastone amino acid group incorporated therein and at least one poly(aminoacid) side chain extending from an amino acid group in the polyesterbackbone. Porous particles having a relatively large mean diameter, forexample greater than 5 μm, can be used for enhanced delivery of atherapeutic agent to the alveolar region of the lung. The porousparticles incorporating a therapeutic agent may be effectivelyaerosolized for administration to the respiratory tract to permitsystemic or local delivery of wide variety of therapeutic agents.

Finally, U.S. Pat. No. 5,019,400 teaches a very low temperature castingof controlled release microspheres The '400 patent describes a processfor preparing microspheres using very cold temperatures to freezepolymer-biologically active agent mixtures into polymeric microsphereswith very high retention of biological activity and material. Polymer isdissolved in a solvent together with an active agent that can be eitherdissolved in the solvent or dispersed in the solvent in the form ofmicroparticles. The polymer/active agent mixture is atomized into avessel containing a liquid non-solvent, alone or frozen and overlayedwith a liquified gas, at a temperature below the freezing point of thepolymer/active agent solution. The cold liquified gas or liquidimmediately freezes the polymer droplets. As the droplets andnon-solvent for the polymer is warmed, the solvent in the droplets thawsand is extracted into the non-solvent, resulting in hardenedmicrospheres.

A disadvantage of the above mentioned techniques when used with proteinsand peptides is that it that proteins and peptides often form aggregateswhen the particle size becomes smaller than about 1 μm, because they areexposed to large vapor-liquid interfaces during water removal. Theseaggregates remain upon reconstitution in buffer. Therefore, suchtechniques may not lead to biologically active micronized proteinpowders.

Furthermore, it is difficult to control the particle size distributionin these processes in many cases. Methods are needed to remove waterfrom solutions of peptides and proteins to produce small particles, withcontrol of the size distribution, without forming protein aggregates.

SUMMARY OF THE INVENTION

The present inventors realized a need for a simple, efficient and robustprocess for freezing either small (<1 mL) quantities of protein solutionor commercial quantities, that can produce stable submicron particles,e.g., protein particles.

More particularly, the present invention includes compositions andmethod for preparing micron-sized or submicron-sized particles bydissolving a water soluble effective ingredient in one or more solvents;spraying or dripping droplets solvent such that the effective ingredientis exposed to an vapor-liquid interface of less than 50, 100, 150, 200,250, 300, 400 or even 500 cm⁻¹ area/volume; and contacting the dropletwith a freezing surface that has a temperature differential of at least30° C. between the droplet and the surface, wherein the surface freezesthe droplet into a thin film with a thickness of less than 500micrometers and a surface area to volume between 25 to 500 cm⁻¹. In oneaspect, the method further includes the step of removing the solventfrom the frozen material to form particles. In one aspect, the dropletsfreeze upon contact with the surface in about 50, 75, 100, 125, 150,175, 200, 250, 500, 1,000 and 2,000 milliseconds. In another aspect, thedroplets freeze upon contact with the surface in about 50 and 150milliseconds. In another aspect, the droplet has a diameter between 2and 5 mm at room temperature. In another aspect, the droplet forms athin film on the surface of between 50 and 500 micrometers in thickness.In another aspect, the droplets have a cooling rate of between 50-250K/s. In another aspect, the particles after solvent removal have asurface area of 10, 15, 25, 50, 75, 100, 125, 150 or 200 m²/gr.

In one embodiment, the effective ingredient is a protein or peptide andthe particle has less than 50% of the peptide or peptide or protein atthe particle surface. The effective ingredient or active agent may aprotein or peptide and the particle has less than 25, 15, 10 or 5% ofthe peptide or peptide or protein at the surface. In another aspect, theparticles are submicron in diameter and may even include particle fibersless than one micron in diameter. In another aspect, the effectiveingredient includes a surfactant peptide or peptide or protein, a DNase,and α-1-antitrypsin, an interleukin, a protease inhibitor, aninterleukin receptor, a monoclonal antibody, a muramyl dipeptide, acatalase, a phosphatase, a kinase, a receptor antagonist, a receptoragonist, a dismutase, a calcitonin, a hormone, an interfereon, insulin,a growth factor, erythropoietin, heparin, vasopressin, peptides,albuterol sulfate, terbutaline sulfate; insulin, glucagon-like peptide,C-Peptide, erythropoietin, calcitonin, human growth hormone, leutenizinghormone, prolactin, adrenocorticotropic hormone, leuprolide, interferonα-2b, interferon beta-1a, sargramostim, aldesleukin, interferon α-2a,interferon alpha, n3 α,-peptide or proteinase inhibitor; etidronate,nafarelin, chorionic gonadotropin, prostaglandin E2, epoprostenol,acarbose, metformin, or desmopressin, cyclodextrin, antibiotics; and thepharmacologically acceptable organic and inorganic salts or metalcomplex thereof.

In one embodiment, the surface is cooled by a cryogenic solid, acryogenic gas, a cryogenic liquid or a heat transfer fluid capable ofreaching cryogenic temperatures or temperatures below the freezing pointof the solvent. In another aspect, the solvent further includes one ormore excipients selected from sugars, phospholipids, surfactants,polymeric surfactants, vesicles, polymers, including copolymers andhomopolymers and biopolymers, dispersion aids, and serum albumin. Inanother aspect, the effective ingredient includes an enzyme and theenzymatic activity of the enzyme is greater than 90%. In another aspect,the effective ingredient includes a peptide or protein and peptide orprotein aggregation is less than 3%. In another aspect, the temperaturedifferential between the droplet and the surface is at least 50° C.

The present invention also includes a pharmaceutical formulation thatincludes drug particles prepared by preparing micron-sized orsubmicron-sized particles by dissolving a water soluble effectiveingredient or active agent in one or more solvents; spraying or drippingdroplets solvent such that the effective ingredient is exposed to anvapor-liquid interface of less than 50 cm⁻¹ area/volume; and contactingthe droplet with a freezing surface that has a temperature differentialof at least 30° C. between the droplet and the surface, wherein thesurface freezes the droplet into a thin film with a thickness of lessthan 500 micrometers and a surface area to volume between 25 to 500cm⁻¹.

Another embodiment of the present invention includes a method forpreparing micron-sized or submicron-sized solvent particles including:spraying or dripping droplets of a water soluble peptide or protein in asolvent, wherein the droplet is exposed to an vapor-liquid interface ofless than 50 cm⁻¹ area/volume; contacting the droplet with a freezingsurface that has a temperature differential of at least 30° C. betweenthe droplet and the surface, wherein the droplet freezes into a thinfilm with a thickness of less than 500 micrometers and a surface area tovolume between 25 to 500 cm⁻¹. The method may further include the stepof removing the solvent from the frozen material to form particles. Inanother aspect, the solvent further includes at least one or moreexcipient or stabilizers selected from, e.g., sugars, phospholipids,surfactants, polymeric surfactants, vesicles, polymers, includingcopolymers and homopolymers and biopolymers, dispersion aids, and serumalbumin. In another aspect, the peptide or protein includes an enzymeand the enzymatic activity of the enzyme is greater than 90%. In anotheraspect, the peptide or protein aggregation is less than 3%. In anotheraspect, the temperature differential between the solvent and the surfaceis at least 50° C. In another aspect, the particle has less than 50% ofthe peptide or protein at the surface. In another aspect, the particlehas less than 25, 15, 10 or 5% of the peptide or protein at the surface.In another aspect, the peptide or protein includes, e.g., a surfactantpeptide or protein, DNase, and α-1-antitrypsin, interleukin, interferon,protease inhibitor, interleukin receptor, monoclonal antibody, muramyldipeptide, catalase, phosphatase, kinase, receptor antagonist, receptoragonist, dismutase, calcitonin, hormone, insulin, a growth factor,erythropoietin, heparin, vasopressin, peptides, glucagon-like peptide,C-Peptide, erythropoietin, human growth hormone, luteinizing hormone,prolactin, adrenocorticotropic hormone, leuprolide, interferon,interferon α-2b, interferon beta-1a, sargramostim, aldesleukin,interferon α-2a, interferon alpha, n3 α,-peptide or proteinaseinhibitor; and the pharmacologically acceptable organic and inorganicsalts or metal complex thereof.

In one embodiment, the present invention includes a formulation, e.g., apharmaceutical formulation or active agent, that includes drug particlesprepared by preparing micron-sized or submicron-sized solvent particlesincluding: spraying or dripping droplets of a water soluble peptide orprotein in a solvent, wherein the droplet is exposed to an vapor-liquidinterface of less than 50 cm⁻¹ area/volume; contacting the droplet witha freezing surface that has a temperature differential of at least 30°C. between the droplet and the surface, wherein the droplet freezes intoa thin film with a thickness of less than 500 micrometers and a surfacearea to volume between 25 to 500 cm⁻¹.

Yet another embodiment includes compositions and methods for preparingmicron-sized or submicron-sized particles by preparing an emulsionincluding a water soluble effective ingredient in solution; spraying ordripping droplets of the solution such that the effective ingredient isexposed to an vapor-liquid interface of less than 50 cm⁻¹ area/volume;and contacting the droplet with a freezing surface that has atemperature differential of at least 30° C. between the droplet and thesurface, wherein the surface freezes the droplet into a thin film with athickness of less than 500 micrometers and a surface area to volumebetween 25 to 500 cm⁻¹.

Yet another embodiment includes a system for preparing solvent nano andmicro-particles that includes a solvent source composed of one or moresolvents; a vessel containing a cryogenic liquid selected from cryogenicliquid selected from the group consisting of carbon dioxide, nitrogen,ethane, propane, helium, argon, or isopentane; and an insulating nozzlehaving an end and a tip, wherein the end of the nozzle is connected tothe solvent source and the tip is placed above, at or below the level ofthe cryogenic liquid. In one aspect, the solution source furtherincludes water, at least one organic solvent, or a combination thereof.In one aspect, the organic solvent is elected from the group consistingof ethanol, methanol, tetrahydrofuran, acetonitril acetone, tert-butylalcohol, dimethyl sulfoxide, N,N-dimethyl formamide, diethyl ether,methylene chloride, ethyl acetate, isopropyl acetate, butyl acetate,propyl acetate, toluene, hexanes, heptane, pentane, and combinationsthereof.

In another embodiment, a method for spray freezing including: spraying asolvent through an insulating nozzle located above, at or below thelevel of a cryogenic liquid, wherein the spray rapidly generates frozensolvent particles having a size range of 10 nm to 10 microns. In oneaspect, the solvent particles produced have a particle size of less than10 microns. In another aspect, the solvent particle has a surface areagreater than 50 m²/g. In one aspect, the cryogenic material is a liquid,a gas, a solid or a surface. In another aspect, the one or more solventscomprises a first solvent that is less volatile than a second solvent,wherein the more volatile solvent is removed but not the second solvent.In yet another aspect, the one or more solvents comprises a firstsolvent that is less volatile than a second solvent, wherein the morevolatile solvent is removed by evaporation, lyophilization, vacuum, heator chemically.

Yet another embodiment of the present invention includes a single-step,single-vial method for preparing micron-sized or submicron-sizedparticles by reducing the temperature of a vial wherein the vial has atemperature differential of at least 30° C. between the solvent and thevial and spraying or dripping solvent droplets of a water solubleeffective ingredient dissolved in one or more solvents directly into thevial such that the effective ingredient is exposed to a vapor-liquidinterface of less than 500 cm⁻¹ area/volume, wherein the surface freezesthe droplet into a thin film with a thickness of less than 500micrometers and a surface area to volume between 25 to 500 cm⁻¹. Thedroplets freeze may upon contact with the surface in about 50, 75, 100,125, 150, 175, 200, 250, 500, 1,000 and 2,000 milliseconds, and may evenfreeze upon contact with the surface in about 50, 150 to 500milliseconds. In one example, a droplet has a diameter between 0.1 and 5mm at room temperature or even a diameter between 2 and 4 mm at roomtemperature. In another example, the droplet forms a thin film on thesurface of between 50 and 500 micrometers in thickness. In one specificexample the droplets will have a cooling rate of between 50-250 K/s. Thevial may be cooled by a cryogenic solid, a cryogenic gas, a cryogenicliquid, a freezing fluid, a freezing gas, a freezing solid, a heatexchanger, or a heat transfer fluid capable of reaching cryogenictemperatures or temperatures below the freezing point of the solvent.The vial may even be rotated as the spraying or droplets are deliveredto permit the layering or one or more layers of the final particles. Inone example, the vial, the water soluble effective ingredient and theone or more solvents are pre-sterilized prior to spraying or dripping.The method may also include the step of spraying or dripping is repeatedto overlay one or more thin films on top of each other to fill the vialto any desired level up to totally full.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1A is a diagram of the thin film freezing process displaying thefalling droplet.

FIG. 1B is a diagram of falling droplet spreading after impact on thestainless steel surface.

FIG. 1C is a diagram of a droplet during cooling and freezing as a thinfilm.

FIGS. 2A and 2B are infrared (IR) photographs of an aqueous dropletimpinging and freezing on a stainless steel surface at 223 K and at 133K, respectively.

FIG. 3 is a plot of IR intensity versus time for an aqueous thin film onstainless steel surface at 223 K.

FIG. 4 is a plot of laser light scattering of particles formed by thinfilm freezing.

FIGS. 5A and 5B are scanning electron micrograph (SEM) images ofparticles from 5 mg/mL lysozyme solutions processed by thin filmfreezing at surface temperatures of 223 K, and 133 K, respectively.

FIG. 5C is a scanning electron micrograph (SEM) of particles from 5mg/mL lysozyme solutions using spray freezing into liquid with liquidnitrogen.

FIG. 6A to 6C are SEM images of particles from 50 mg/mL lysozymesolution processed by thin film freezing, by spray freezing into liquidnitrogen, and by spray freeze-drying-10 μm into liquid nitrogen,respectively.

FIG. 7A is a graph of temperature versus depth profiles of thin aqueousfilms cooled on a surface at 223 K for a 220 μm thin film.

FIG. 7B is a graph of temperature versus depth profiles of thin aqueousfilms cooled on a surface at 133 K for a 320 μm thin film.

FIG. 8A is a picture of nucleation and growth of protein particle inunfrozen channels between glassy frozen water domains with highsupercooling in the thin film freezing, spray freezing into liquid, andspray freeze-drying processes.

FIG. 8B is a picture of nucleation and growth of protein particle inunfrozen channels between glassy frozen water domains with lowsupercooling in shelf lyophilization.

FIG. 9 is a graph of freezing time versus exposure to gas-liquidinterface for lyophilization, thin film freezing (TFF), spray freezinginto liquid (SFL), and spray freeze-drying (SFD).

FIG. 10A is a SEM image of top of dried lysozyme thin film at thecenter.

FIG. 10B is a SEM image of top of dried lysozyme thin film atapproximately 10 μm from the edge.

FIG. 11 is a graph that shows thin film freezing of lysozyme withvarious amounts of Ethanol in original Concentration Measured after 10minutes of sonication by Malvern Mastersizer.

FIG. 12 is a graph that shows various high initial solubilizedconcentrations of lysozyme frozen by TFF and then lyophilized.

FIGS. 13A and 13 B shows the morphologies of TFF lysozyme prepared inglass vial (TFF lys: feed=5 mg/mL prepared directly in a glass vial)versus a TFF lysozyme prepared on a drum (FIGS. 13C to 13D)(TFF lys:feed=5 mg/mL prepared on TFF drum).

FIG. 14 shows the resuspended suspension of TFF particles in a suitablesolvent for parenteral delivery.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

The ability to produce high surface area stable submicron andmicron-sized protein particles would create new opportunities for oral,depot, pulmonary, and transdermal delivery applications (1-9). Inpulmonary delivery, high surface area porous particles with aerodynamicdiameters between 1-3 μm may be deposited more efficiently in the deeplung compared to dense particles with similar aerodynamic diameters (1,8). In depot delivery, 300-500 nm submicron protein particles have beenencapsulated uniformly into 10-50 μm diameter microspheres to achievehigh protein loadings, while minimizing burst release (4, 6, 10, 11).

Solid protein particles, stabilized by cryoprotectants including sugars,are often less susceptible to destabilization during storage (1, 12-17)relative to proteins in solution. However, the formation of stablesubmicron protein particles with surface areas exceeding 10 m²/g (4, 18,19) is highly challenging, as the removal of water exposes proteinmolecules to large interfacial areas. Adsorption of protein atgas-liquid and ice-liquid interfaces often results in unfolding andaggregation (1, 18-22). In lyophilization, the most common process forproducing stable protein particles, particle growth during slow cooling(˜1 K/min) limits the particle diameter to a minimum of a few micronswith surface areas less than 1 m²/g (21). The same limitation is truewhen drop freezing small aliquots (˜20-50 μL) of protein solution intoliquid nitrogen (23), freezing thick (>500 μm) films on a cooled shelf(24), and plunge freezing ultra-thin walled PCR tubes filled withprotein solution into liquid nitrogen (23). In these techniques, theprotein solution was cooled at rates between 1 to 10 K/s (23, 24).Although the dried particles may be milled to form submicron particles,yields can be limited, size distributions are often broad, and themechanical stress can lead to denaturation (1, 21). Submicron proteinparticles may be precipitated from aqueous solution by a variety ofprocesses including spray-drying (11, 21, 22, 25), supercriticalCO₂-assisted aerosolization and bubble drying (scCO₂A-BD) (26), sprayfreeze-drying (SFD) (1, 18, 19, 21), and spray freezing into liquids(SFL).

As used herein, “bioavailability” is a term meaning the degree to whicha drug becomes available to the target tissue after being administeredto the body. Poor bioavailability is a significant problem encounteredin the development of pharmaceutical compositions, particularly thosecontaining an active ingredient that is not highly soluble in water. Incertain embodiments, the proteins may be water soluble, poorly soluble,not highly soluble or not soluble. The skilled artisan will recognizethat various methodologies may be used to increase the solubility ofproteins, e.g., use of different solvents, excipients, carriers,formation of fusion proteins, targeted manipulation of the amino acidsequence, glycosylation, lipidation, degradation, combination with oneor more salts and the addition of various salts.

As used herein, the term “effective ingredient” refers to a compound orcompounds, whether in pure or partially purified form (e.g., extracts)that has an known effect on target. For example, pharmaceutical agentsare effective ingredients for their known target, e.g., penicillin is aneffective ingredient or agent against susceptible bacteria. Anotherexample of an effective ingredient is an insecticide that has a knowninsect target. The present invention may be used to manufacture anddelivery effective ingredients against targets in a manner that willenhance its delivery, as specifically described hereinbelow.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

Particle formation technologies may be classified as either mechanicalmicronization processes or solution-based phase separation processes.Mechanical micronization methods include milling techniques such as thatcited in U.S. Pat. No. 5,145,684. However, friction generated duringthese milling processes may lead to either thermal or mechanicaldegradation of the active pharmaceutical ingredient. Spray drying,another common method used to micronize drug substances, requiresextremely high temperatures, on the order of 150° C., to remove thesolvent from the drug following atomization. The elevated temperaturesmay accelerate degradation of the active ingredient.

Non-limiting examples of effective ingredients are pharmaceuticals,pharmaceutical agents, peptides, nucleic acids, proteins, antibiotics,gene therapy agents, catalysts, adsorbents, pigments, coatings, personalcare products, abrasives, particles for sensors, metals, alloys,ceramics, membrane materials, nutritional substances, anti-canceragents, as well as, chemicals used in the agriculture industries such asfertilizers, pesticides and herbicides. It will be appreciated that thislist is not exhaustive and is for demonstrative purposes only. It willbe further appreciated that it is possible for one compound to beincluded in more than one class of effective ingredients, for example,peptides and pharmaceuticals.

Examples of effective ingredients that are pharmaceutical agentsinclude, but are not limited to, antibiotics, analgesics,anticonvulsants; antidiabetic agents, antifungal agents, antineoplasticagents, antiparkinsonian agents, antirheumatic agents, appetitesuppressants, biological response modifiers, cardiovascular agents,central nervous system stimulants, contraceptive agents, diagnosticagents, dopamine receptor agonists, erectile dysfunction agents,fertility agents, gastrointestinal agents, hormones, immunomodulators,antihypercalcemia agents, mast cell stabilizers, muscle relaxants,nutritional agents, ophthalmic agents, osteoporosis agents,psychotherapeutic agents, parasympathomimetic agents, parasympatholyticagents, respiratory agents, sedative hypnotic agents, skin and mucousmembrane agents, smoking cessation agents, steroids, sympatholyticagents, urinary tract agents, uterine relaxants, vaginal agents,vasodilator, anti-hypertensive, hyperthyroids, anti-hyperthyroids,anti-asthmatics and vertigo agents. Further examples of effectiveingredients include a cardiovascular drug, respiratory drug,sympathomimetic drug, cholinomimetic drug, adrenergic or adrenergicneuron blocking drug, antidepressant, antihypertensive agent,anti-inflammatory, antianxiety agent, immunosuppressive agents,antimigraine agents, sedatives/hypnotic, antianginal agents,antipsychotic agents, antimanic agents, antiarrhythmic, antiarthriticagent, antigout agents, anticoagulant, thrombolytic agents,antifibrinolytic agents, hemorheologic agents, antiplatelet agents,anticonvulsant, antihistamine/antipruritic, agent useful for calciumregulation, antiviral agents, anti-infective, bronchodialator, hormone,hypoglycemic agent, hypolipidemic agent, protein, nucleic acid, agentuseful for erythropoiesis stimulation, antiulcer/antireflux agent,antinauseant/antiemetic, oil-soluble vitamin, mitotane, visadine,halonitrosourea, anthrocycline or ellipticine.

The pharmaceutical effective ingredients may be used in a variety ofapplication modalities, including oral delivery as tablets, capsules orsuspensions; pulmonary and nasal delivery; topical delivery asemulsions, ointments or creams; and parenteral delivery as suspensions,microemulsions or depot. The resulting powder can be redispersed at anyconvenient time into a suitable aqueous medium such as saline, bufferedsaline, water, buffered aqueous media, solutions of amino acids,solutions of vitamins, solutions of carbohydrates, or the like, as wellas combinations of any two or more thereof, to obtain a suspension thatcan be administered to mammals.

The solution agent used in the solution can be an aqueous such as water,one or more organic solvents, or a combination thereof. When used, theorganic solvents can be water soluble or non-water soluble. Suitableorganic solvents include but are not limited to ethanol, methanol,tetrahydrofuran, acetonitrile, acetone, tert-butyl alcohol, dimethylsulfoxide, N,N-dimethyl formamide, diethyl ether, methylene chloride,ethyl acetate, isopropyl acetate, butyl acetate, propyl acetate,toluene, hexanes, heptane, pentane, and combinations thereof.

The excipients and adjuvants that may be used in the present invention,while potentially having some activity in their own right, for example,antioxidants, are generally defined for this application as compoundsthat enhance the efficiency and/or efficacy of the effectiveingredients. It is also possible to have more than one effectiveingredient in a given solution, so that the particles formed containmore than one effective ingredient.

As stated, excipients and adjuvants may be used to enhance the efficacyand efficiency of the effective ingredients. Non-limiting examples ofcompounds that can be included in the solutions that are to be sprayfrozen in accordance with the present invention include:cryoprotectants, lyoprotectants, surfactants, fillers, stabilizers,polymers, protease inhibitors, antioxidants and absorption enhancers.The excipients may be chosen to modify the intended function of theeffective ingredient by improving flow, or bio-availability, or tocontrol or delay the release of the effective ingredient. Specificnonlimiting examples include: sucrose, trehaolose, Span 80, Tween 80,Brij 35, Brij 98, Pluronic, sucroester 7, sucroester 11, sucroester 15,sodium lauryl sulfate, oleic acid, laureth-9, laureth-8, lauric acid,vitamin E TPGS, Gelucire 50/13, Gelucire 53/10, Labrafil, dipalmitoylphosphadityl choline, glycolic acid and salts, deoxycholic acid andsalts, sodium fusidate, cyclodextrins, polyethylene glycols, labrasol,polyvinyl alcohols, polyvinyl pyrrolidones and tyloxapol. Using theprocess of the present invention, the morphology of the effectiveingredients can be modified, resulting in highly porous microparticlesand nanoparticles.

In certain embodiments, the present invention demonstrates a novelmethod to produce stable submicron protein particles. The method isherein referred to as thin film freezing (TFF). FIG. 1 illustrates oneembodiment of TFF. In TFF, liquid droplets typically fall from a givenheight and impact, spread, and freeze on a cooled solid substrate. InFIG. 1A, a droplet 10 falls from a given height, and impact a spinningsurface 12 that has a temperature of 223 K. As the droplet spreads out,freezing front 14 is formed in advance of the unfrozen liquid 16 shownin FIG. 1B. Typically, the size of the completely frozen droplet 18 isabout 12 mm in diameter, with a height approximently 216 μm. Recently,TFF was used to form high SSA powder (25-29 m²/g) of the poorly watersoluble drug danazol (45). Liquid droplets (˜2-4 mm in diameter) weredispensed from a pipet above a cryogenically cooled metal surface (46,47). Upon impact, the droplets spread out into thin films (˜100-400 μm)that froze on time scales of 70 to 1000 ms, which corresponds to acooling rate of ˜10² K/s (42-44, 47-57). The cooling rates predictedwith a 1-D heat transfer model were in agreement with laboratorymeasurements with an infrared (IR) camera (45). Since the cooling ratesin TFF and SFL are comparable, TFF may be expected to be a desirableprocess for forming high surface area protein particles.

As will be apparent to those of skill in the art, the droplets may bedelivered to the cold or freezing surface in a variety of manners andconfigurations. For example, to provide for high-throughputcapabilities, the droplets may be delivered in parallel, in series, atthe center, middle or periphery or a platen, platter, plate, roller,conveyor surface. The freezing or cold surface may be a roller, a belt,a solid surface, circular, cylindrical, conical, oval and the like thatpermit for the droplet to freeze. For a continuous process a belt,platen, plate or roller may be particularly useful. In operation, frozendroplets may form beads, strings, films or lines of frozen substrate andeffective ingredient that are removed from the surface with a scraper,wire, ultrasound or other mechanical separator prior to thelyophilization process. Once the material is removed from the surface ofthe belt, platen, roller or plate the surface is free to receiveadditional material in a continuous process.

In certain embodiments, the present invention demonstrate submicron LDHand lysozyme particles (>10 m²/g) with 100% enzyme activity may beformed with TFF followed by lyophilization. The cooling rate wasdesigned to be sufficiently fast to arrest particle growth, whereas therelatively small liquid-gas interfacial surface area helps preventprotein adsorption, unfolding and aggregation. The present inventionpresents dimensions of the thin films, stabilities (enzyme activity) ofLDH powders after reconstitution, and morphologies of lysozyme particlesdetermined by SEM and BET measurements of surface area. The presentinvention also gives cooling rates of the thin films determined by a 1-Dheat transfer model and IR measurement. The cooling rates, particlemorphologies and protein stabilities for the intermediate cooling rateprocesses TFF and SFL, relative to the ultra-rapid cooling process, SFD,and in the slow process, lyophilization were also compared. A proteinnucleation and growth mechanism is presented to illustrate the particlemorphologies in terms of the cooling rates. In TFF, the much smallerarea of the gas-liquid interface of the falling droplet and spread filmrelative to the atomized droplets in SFD is shown to result insignificantly less protein adsorption, and consequently, minimaldenaturation and aggregation. Furthermore, the intermediate cooling rate(˜10² K/s) is shown to be sufficient to arrest particle growth to givesurface areas >30 m²/g.

Compared to SFD and SFL, TFF offers the advantage of simplification inthe processing steps, in addition to improvement in the stability of theprotein. TFF on a cold metal surface bypasses the need to maintainaseptic conditions of a liquid cryogen, for example liquid nitrogen, inthe SFD and SFL processes (24). The cooling rate of the thin films inTFF may be controlled readily by varying the temperature of the metalsurface. Also, the surface temperature of the film may be measureddirectly (45). For SFL and SFD, the complex geometry of the turbulentspray in the liquid nitrogen (LN₂) combined with the Leidenfrost effectcan be somewhat difficult to control and monitor (36). In TFF, moreconcentrated and thus more viscous solutions may be processed, as thedroplets do not need to be atomized. In TFF, collection of the frozenfilms leads to nearly 100% yields. However, in SFD process yields wereonly about 80% as the result of entrainment of uncaptured particles inthe atomized aqueous stream, particles sticking to the sides ofcollection vessels, and inefficient separation of the cryogen from the10-100 μm frozen particles (11, 21).

Materials. Lysozyme was purchased from Sigma and L-LDH from porcineheart suspended in a 3.2 M ammonium sulfate solution from Roche AppliedScience. Trehalose, NADH and pyruvate were purchased from Sigma. Thewater was deionized by flowing distilled water through a series of 2×7 Lmixed bed vessels (Water and Power Technologies) containing 60:40anionic:cationic resin blends.

LDH Enzyme preparation and catalytic activity assay. The LDH enzymepreparation and catalytic activity assay used in the present inventionis described in detail in a previous reference (32). The LDH in ammoniumsulfate was dialyzed against 10 mM KPO₄ buffer (pH 7.5) at 4° C. for 3hours before use (58, 59). LDH activities were measured for the reactionof pyruvate and NADH into lactate and NAD+. Units of LDH activity (U)were calculated by measuring the decrease in absorbance of NADH at λ=340nm every 15 seconds for 1 minute due to the conversion of NADH to NADover time (U=Δμmol NADH/min) and then dividing by the mass (mg) of theLDH protein in solution to determine specific activity (U/mg). Thestability of the LDH formulation in 30 mg/mL trehalose was measured overtime. The LDH specific activity remained stable for an hour and thenbegan to decrease. All results were performed in the time period wherethe LDH specific activity had not decayed. During this time period, thespecific activity was defined as 100%.

Example of the Thin Film Freezing (TFF) procedure. Aqueous proteinsolutions of LDH or lysozyme were passed at a flow rate of 4 mL/mineither through a 17 gauge (1.1 mm ID, 1.5 mm OD) stainless steel syringeneedle producing 3.6 mm diameter droplets or through 3.9 mm ID, 6.4 mmOD stainless steel tubing producing 5.6 mm diameter droplets. Thedroplets fell from a height of 10 cm above a rotating stainless steeldrum 17 cm long and 12 cm in diameter. The stainless steel drum washollow with 0.7 cm thick walls and was filled with dry ice or liquidnitrogen to maintain drum surface temperatures of 223 K or 133 K,respectively. Before each run, the surface temperature of the drum wasverified with a DiGi-Sense® Type K thermometer using a 45° angle surfaceprobe thermocouple attachment (Eutech Instruments). The drum rotated atapproximately 12 rpm and was powered by a Heidolph RZR2041 mechanicaloverhead stirrer (ESSLAB) connected to a speed reducer. On impact thedroplets deformed into thin films (FIG. 1) and froze. The frozen thinfilms were removed from the drum by a stainless steel blade mountedalong the rotating drum surface. The frozen thin films then fell 5 cminto a 400 mL Pyrex® beaker filled with liquid nitrogen. For lysozyme,the frozen thin films in the 400 mL Pyrex® beakers were transferreddirectly to a −80° C. freezer to evaporate excess liquid nitrogen. ForLDH, the frozen thin films were transferred from the 400 mL Pyrex®beakers into 50 mL polypropylene tubes (Part No. UP2255, UnitedLaboratory Plastics) 2 cm in diameter and 16 cm in height using aspatula pre-cooled in liquid nitrogen.

Infrared Imaging of Cooling Thin Films. An InSb focal plane array (FPA)camera (Phoenix digital acquisition system (DAS camera, Indigo Systems)was positioned to acquire infrared images from above the cooling thinfilm on a flat plate. The FPA camera detected 3-5 μm radiation, and theimages were acquired at 100 frames per second (10 ms/image). Thedimensions of each frame were 256 pixels by 256 pixels (15 mm×15 mm).The image spatial resolution was approximately 40 μm per pixel. Averageintensity values were calculated using MATLAB® version 6 (20×20 pixelsquare within the center of the droplet) and plotted versus time todetermine the time for the center of the thin film to reach thermalequilibrium with the plate.

Drying and shelf loading. A Virtis Advantage Lyophilizer (The VirtisCompany, Inc.) was used to dry the frozen slurries. The 400 mL beakerscontaining frozen slurries of lysozyme and the 50 mL polypropylene tubescontaining the frozen slurries of LDH were covered with a single layerKim-wipe. Primary drying was carried out at −40° C. for 36 hrs at 300mTorr and secondary drying at 25° C. for 24 hrs at 100 mTorr. A 12 hourlinear ramp of the shelf temperature from −40° C. to +25° C. was used at100 mTorr.

LDH reconstitution and concentration assay. Dried LDH powders werereconstituted with 1 mL of DI water and the enzyme assay was performedimmediately. After all protein samples had been analyzed for enzymaticactivity, the protein concentration was measured with the BCA(bicinchoninic acid) protein analysis kit (Sigma Chemical Company). Onceprotein concentrations were determined, the specific activity from eachmeasurement could be calculated. The activity of each LDH sample wasnormalized by the specific activity of the control measured immediatelybefore the freezing process.

Transfer and storage of dried powders. After the lyophilization cyclewas complete, the lyophilizer was purged with nitrogen upon releasingthe vacuum to reduce the exposure time of the protein powders to watervapor in the ambient air before transfer. The samples were then rapidlytransferred to a dry box held at 14% RH, and the powders weretransferred to 20 mL scintillation vials. The vials were then coveredwith 24 mm Teflon® Faced Silicone septa (Wheaton) which were held inplace by open-top screw cap lids. Vials were purged with dry nitrogenfor 2 minutes via a needle through the septa and an additional needlefor the gas effluent.

Surface area measurement. Surface areas of dried powders were measuredwith a Quantachrome Nova 2000 (Quantachrome Corporation) BET apparatus.Dried powders were transferred to the glass BET sample cells in a drybox. Samples were then degassed under vacuum for a minimum of 12 hours.The Brunauer, Emmett, and Teller (BET) equation (60) was used to fitadsorption data of nitrogen at 77 K over a relative pressure range of0.05-0.30. The samples were measured two times.

Residual moisture content. Aliquots of methanol were dispensed throughthe septum of the scintillation vials to form a suspension concentrationof 10-100 mg/mL. Vials were then placed in a bath sonicator (MettlerElectronics) for 5 minutes at maximum power to insure completesuspension of the powder. Moisture content was measured for a 200 μLaliquot with an Aquatest 8 Karl-Fischer Titrator (PhotovoltInstruments). The moisture values were corrected with a 200 μL methanolblank control. All samples had a moisture content between 6-8% (w/w)after drying, comparable to values of 2-7% (w/w) for BSA prepared by SFD(18).

Particle size analysis. The size distribution of dried powders wasmeasured by multiangle laser light scattering with a MalvernMastersizer-S (Malvern Instruments). A mass of 30-100 mg of powder wassuspended in 10 mL of acetonitrile and the suspension was then sonicatedon ice for 1 minute using a Branson Sonifier 450 (Branson UltrasonicsCorporation) with a 102 converter and tip operated in pulse mode at 35W. Typical obscuration values ranged from 11% to 13%. Aliquots of thesonicated suspension were then dispensed into a 500 mL acetonitrile bathfor analysis.

Scanning electron microscopy (SEM). SEM images were collected on aHitachi Model S-4500 scanning electron microscope (Hitachi Ltd). Thesamples were prepared in a dry-box. Aluminum stages fitted with doubleadhesive carbon conducting tape were gently dipped into sample vialsuntil covered by powder. Stages were then placed in septum capped vialsand purged with nitrogen for transfer. To minimize the time samples wereexposed to atmospheric moisture the stages were rapidly transferred to aPelco Model 3 sputter-coater. A conductive gold layer was applied andthe samples were then quickly transferred to the SEM. Total exposure tothe atmosphere was less than 1 minute.

Table 1 (below) shows the characterization of thin films formed fromdeionized water droplets as a function of surface temperature anddroplet diameter.

TABLE 1 Thin Film from Thin Film from 3.6 mm Drop^(b) 5.6 mm Drop^(c)SFD^(a) SFL^(a) 223 K^(d) 133 K^(d) 223 K^(d) 133 K^(d) Droplet or 10100 12000 10000 23000 19000 Thin Film Disk Diameter (μm) Film Thickness— — 216 311 221 324 (μm) Droplet or 6000 600 46 32 45 31 Thin FilmSurface Area to Volume (cm⁻¹) ^(a)Values taken from Engstrom et al. (36)^(b)Surface Area to Volume of 3.6 mm droplet is 17 cm⁻¹ ^(c)Surface Areato Volume of 5.6 mm droplet is 11 cm⁻¹ ^(d)Temperatures of stainlesssteel plate

The droplets spread on the cold metal surface and formed a cylindricalthin disk. The disk diameter decreased with a decrease in surfacetemperature from 223 K to 133 K and increased with an increase infalling droplet radius. Since the frozen thin films were cylindricaldisks, the thicknesses of the thin films were calculated from the knownvolume of the liquid droplet and the measured disk diameter. The volumesof the falling droplets were determined by counting the number ofdroplets required to occupy 1 mL in a graduated cylinder. The averagethin film thickness for the 223 K and 133 K surfaces were 220 μm and 320μm, respectively. The corresponding surface area/volume ratios for thetop surfaces of the cylinders are also shown in Table 1. The filmthicknesses were essentially independent of the falling dropletdiameter. For aqueous samples containing concentrations of lysozymebetween 5 and 50 mg/mL or trehalose at 30 mg/mL, the droplet volumes,disk diameters, and thus film thicknesses did not change relative topure water. The surface area/volume ratios for the 3.6 mm and 5.6 mmfalling droplets in TFF were 17 cm⁻¹ and 11 cm⁻¹, respectively. As shownin Table 1, upon impact, the falling droplets spread into thin filmswith final surface area/volume ratios between 31 and 46 cm⁻¹. In aprevious reference (36) of SFD and SFL, the corresponding surfacearea/volume ratios were 6000 and 600 cm⁻¹, respectively. Relative tothese values, the much smaller surface area/volume ratio for TFF may beexpected to lower the degree of protein destabilization from exposure tothe gas-liquid interface.

The thin films were further characterized by determining the coolingrates from infrared measurements. The IR camera outputs intensity valueswith white indicating a high intensity and black a low intensity inrelation to the amount of radiant energy E (energy density per unit timeper unit wavelength) emitted from the droplet (45, 61). The radiantenergy E is related to the temperature of the object according toPlanck's law as equation (1)

E(λ,T)=(2πhc ²)/{λ⁵[exp(hc/λkT)−1]}  Equation (1)

where λ is the wavelength, c is the speed of light, k is the Boltzmannconstant, h is Planck's constant and T is the temperature in Kelvin(61). Therefore, the intensity output of the IR camera is relateddirectly to the temperature.

For the thin film on the 223 K surface shown in FIG. 2A, the diameter ofthe film was 12 mm and the edge was uniform and smooth. As coolingprogressed, a cooling front moved radially inward from the edge of thefilm toward the center. The center of the film reached thermalequilibrium in 1.6 s shown in FIG. 2A and FIG. 3. For the thin film onthe 133 K surface demonstrated in FIG. 2B, the diameter was 10 mm anddark jagged “fingers” were observed at the edge, indicating the coldestdomains. The cooling front moved radially inward from the edge to thecenter at first. Next, the center turned black, and an annular regionbetween the center and the outer jagged edge remained gray. The coolingfront then reversed direction by moving from the center toward the edgeof the film. FIG. 3 shows the center of the film reached thermalequilibrium a little more slowly, in about 3 s, relative to 223 K. Ineach case at the center of the film, a plateau was observed and then anabrupt final decay to thermal equilibrium.

The LDH activities for an aqueous formulation of 0.25 mg/mL LDH with 30mg/mL trehalose frozen by lyophilization, SFL (32), and TFF wereextremely high and not significantly different (p<0.05) according to aStudent's t test shown in Table 2. Table 2 shows activities for 0.25mg/mL LDH, 30 mg/mL trehalose formulations frozen by various techniquesin pH 7.5, 10 mM KPO₄ buffer in replicates of 3.

TABLE 2 % Activity Freezing Process 223 K 133 K Thin Film (3.6 mm drop)100 ± 3.9 104 ± 12.0 Thin Film (5.6 mm drop)  97 ± 9.5 100 ± 8.4 SFL^(a,d) 98 ± 5.3 SFD-130 μm^(a) 85 ± 8.2 SFD-40 μm^(a,e) 74 ± 6.7SFD-10 μm^(a,d) 80 ± 5.4 Falling Droplet (3.6 mm)^(c) 98 ± 2.1 Sprayinto Air (10 μm)^(a,b,c) 85 ± 7.7 Lyophilization 99 ± 2.1 ^(a)Valuestaken from Engstrom et al. (32) ^(b)100 mg/mL trehalose used in LDHformulation ^(c)The droplets were not frozen in these two controls^(d)Replicate of 4 ^(e)Replicate of 5

Compared to the SFD process for three droplet sizes, the LDH activitiesfor each TFF condition were significantly higher (p<0.05). The very highLDH activities were maintained in the TFF process throughout the serialstresses of droplet falling and spreading, freezing, drying, andreconstitution.

Given the high enzyme activities for LDH particles formed by TFF, theother key goal was to demonstrate particle morphologies with submicronparticle sizes and large particle surface areas. Table 3 demonstratesspecific surface area measurements and particle size distributions forlysozyme powders formed by thin film freezing, SFL, and SFD.

TABLE 3 Lysozyme Concentration SSA (m²/g) Size (μm) Freeze Process(mg/mL) 223 K^(a) 133 K^(a) 223 K^(a) 133 K^(a) Thin Film (3.6 5 73 ±0.8 45 ± 0.4 0.050-1.0 (88%) 0.050-1.0 (81%) mm drop)   1.0-10 (12%)  1.0-12 (19%) Thin Film (5.6 5 — — 0.050-1.0 (92%) 0.050-1.0 (84%) mmdrop)   1.0-12 (8%)   1.0-10 (16%) Thin Film (3.6 50 31 ± 0.1 55 ± 0.40.050-1.0 (66%) 0.050-1.0 (62%) mm drop)   1.0-30 (34%)   1.0-30 (38%)SFL^(b) 5 114 ± 11 0.050-1.0 (85%)   2.0-10 (15%) SFL^(b) 50 34 ± 20.050-1.0 (48%)   4.0-12 (52%) SFD-10 μm^(b) 50 126 ± 5  0.050-1.0 (74%)  1.0-10 (26%) Lyophilization 5  4.4 ± 0.2 0.05-1.0 (7%)   4.8-120 (93%)^(a)Temperatures of stainless steel plate ^(b)Values taken from Engstromet al. (36)

In the case of LDH, the ratio of LDH:trehalose was 1:120 by mass. Asdiscussed previously (32, 36), the particle surface area for trehalosedecreased upon exposure to atmospheric moisture which lowers the Tgsharply. (This limitation may be overcome in the future with the use oflyostoppers to seal the vials from moisture.) Thus, we chose lysozyme asa model protein to investigate powder morphology instead ofLDH:trehalose. Lysozyme samples obtained and transfered at roomtemperature had moisture contents between 6-8% as determined by KarlFischer titration. For moisture contents between 7-8% by weight, the Tgremained high, between 50-60° C. (62). Therefore the loss in lysozymeSSA during transfer may be expected to be negligible. For most cases,the SSA values were similar ranging between 30 and 55 m²/g. For 5 mg/mLlysozyme, the thinner films at 223 K produced a significantly higher SSAof 73 m²/g relative to the films at 133 K. In a previous reference (36),5 and 50 mg/mL lysozyme solutions processed by SFL had measured powderSSAs of 114 m²/g and 34 m²/g, respectively, similar to the valuesproduced by TFF (36). Although the SSA of 126 m²/g for SFD was about 2fold larger than for TFF, the enzyme activity was much smaller, as shownin Table 2.

As shown in Table 3, the volume percentage of submicron particles,determined by laser light scattering, after sonication of the 5 mg/mLlysozyme formulation prepared by TFF at 223 K, ranged from 88 to 92%.The similarity in these two values was expected since the nearlyidentical thin film thicknesses would be expected to produce similarcooling rates. These values were similar to those for the SFL powders(36). For TFF, the protein powders were friable and could be broken upreadily into submicron particles with minimal sonication. As shown inFIG. 4, the D(v,50) was 300 nm. In contrast, the same 5 mg/mL lysozymeformulation prepared by lyophilization had a very low fraction of 7%submicron particles shown in FIG. 4 and Table 3. As the lysozyme feedconcentration was raised to 50 mg/mL the submicron fraction decreased to66 and 62% on the 223 K and 133 K surfaces, respectively. Thecorresponding value for SFL was lower (48%), whereas for SFD it washigher (74%) (36). For the SFD powders, the D(v,50) was approximately300 nm (36). A second peak with micron-sized particles was present forthe 50 mg/mL lysozyme solution prepared by SFL and TFF as shown in Table3. However, 50 mg/mL is an unusually high protein concentration and TFFwould ordinarily be applied to concentrations on the order of 5 mg/mL,where the second larger peak is not present as shown in FIG. 4.

Selected SEM images from the results in Table 3 are shown in FIGS. 5 and6. For 5 mg/mL lysozyme, fine 50 nm primary particles were produced byTFF at 223 K, demonstrated in FIG. 5A, comparable to those produced bySFL (36) in FIG. 5C. At 133 K, larger 50-100 nm diameter particles weremixed with rods 50-100 nm in diameter and more than 500 nm long as seenin FIG. 5B. The larger particles sizes shown in FIG. 5B compared to FIG.5A are consistent with the slightly lower content of submicron particlesmeasured by light scattering listed in Table 3.

For highly concentrated 50 mg/mL lysozyme solutions and a surfacetemperature of 223 K, large sheets were observed with features between 1and 2 μm as shown in FIG. 6A. Similar features were observed for SFL(36). In contrast, a fine web with 100 nm features were produced by SFD(36) seen in FIG. 6C, which is consistent with the smaller particlesizes measured by light scattering in Table 3. The larger featuresobserved in the TFF and SFL processes for 50 mg/mL versus 5 mg/mLsolutions are consistent with the particle size distributions measuredby light scattering. The similarity of the particle morphologies for thepowders prepared by the SFL and TFF processes at both the 5 and 50 mg/mLconcentrations are also examined in terms of cooling rates.

Modeling the cooling rate of thin films. Droplet spreading to form thinfilms of liquid metal and water droplets has been described in term ofthe Weber number, (inertial to interfacial forces) where is the impactvelocity, is the droplet diameter, and is the droplet interfacialtension in air. For We>30 immediately before impacting the cooled solidsubstrate (42, 48-50, 56, 63) the droplets deformed into cylindricalthin films before freezing. For the low We<1 regime, impacting dropletsfroze as spherical domes with minimal droplet spreading (49, 64). Forthe falling liquid droplets, γ(air-water)=72 mN/m and V=(2 gH)^(1/2)(65) where the falling height, H, of the droplet was 10 cm, resulting inV=1.4 m/s. The observed formation of thin cylindrical disks wasconsistent with this We of 97, but when H was reduced to less than 1 cm(We=9.8) the impacting water droplets froze as spherical domes that wereonly 4 mm in diameter.

Previously, it was shown with IR imaging studies of thin films formedwith acetonitrile and t-butanol that droplet spreading occurred withinthe first 10 ms interval indicating that the droplet spreading time wasmuch less than the freezing time (45). The same behavior was observed inFIG. 2 for water. The prediction of the cooling rate of the film with asimplified analytical heat transfer model was in good agreement withlaboratory produced IR data (45). Herein, this approach is extended tothin film freezing of water droplets.

Briefly, the model assumes that the droplet spreads to form acylindrical film on a much shorter time scale than heat transfer. Sincethe height (thickness) of the thin film is on the order of 200-400 μm,relative to a much larger diameter of 10-12 mm, radial heat transfer isneglected. The thermal diffusivity, α=k/ρ*C_(p), where k is the thermalconductivity, ρ is the density, and C_(p) is the heat capacity, istreated as constant over the entire temperature range. For the case offreezing water the thermal diffusivities of water and ice are averaged.One-dimensional heat transfer for a finite slab with an insulatingboundary condition on the top surface of the thin film (air) and aconstant temperature boundary condition on the bottom is described byequation (2)(66):

$\begin{matrix}{{T\left( {x,t} \right)} = {T_{p} + {\frac{2}{L} {\sum\limits_{n = 0}^{\infty}\; {^{{- {\alpha {({{2n} + 1})}}^{2}}\pi^{2}{t/4}L^{2}}\cos  \frac{\left( {{2n} + 1} \right)\pi \; x}{2L}\begin{Bmatrix}{\frac{2{L\left( {- 1} \right)}^{n + 1}T_{p}}{\left( {{2n} + 1} \right)\pi} +} \\{\int_{0}^{L}{T_{i}\frac{{\cos \left( {{2n} + 1} \right)}\pi \; x^{\prime}}{2L}\ {x^{\prime}}}}\end{Bmatrix}}}}}} & {{Equation}\mspace{14mu} (2)}\end{matrix}$

where x is the distance from the top of the spread droplet, T is thetemperature in the film, T_(p) is the plate temperature in contact withthe bottom thin film surface, and L is the film thickness.

The calculated temperature profiles from equation (2) are shown in FIG.7 and the calculated cooling rates and times are shown in Table 4 wherecalculated cooling rates, cooling times, and exposure time to thegas-liquid interface for SFD, SFL, and TFF are listed. The dropletdimensions are given in Table 1.

TABLE 4 Thin Film Thin Film from from 3.6 3.6 mm Drop mm Drop SFD^(a)SFL^(a) 223 K 133 K Cooling Rate (K/s) 3.8 × 10⁶ 7.2 × 10³ 3.9 × 10² 2.0× 10² Cooling Time (ms) 0.033 17 2.0 × 10² 6.2 × 10² Droplet Gas-Liquid10-1000 2 ~1000 ~1000 Exposure Time (ms) ^(a)Values taken from Engstromet al. (36)

The cooling time was defined as the time for the temperature of the topsurface of the film, T(0,t), to decrease from room temperature (25° C.)to a value 5% greater than that of the metal surface. The cooling rate(K/s) was then determined by dividing the temperature difference at thetop of the film by the cooling time. As shown in FIG. 7A and Table 4,the predicted time to cool the top surface of the 220 μm thick thin filmon the 223 K surface is 2.0×10² ms (cooling rate of 3.9×10² K/s). Thecalculated cooling rate is an order of magnitude less than for SFL(7.2×10³ K/s) and 4 orders of magnitude less than for SFD (3.8×10⁶ K/s).The much smaller cooling rates in TFF versus SFD may be explained by a100 fold smaller surface area/volume ratio and a film thickness on theorder of 20-30 times larger than the droplet radius in SFD.

The particle morphologies shown in FIG. 5 and particle SSAs in Table 3were similar for freezing on the 223 K and 133 K surfaces, as aconsequence of the rapid cooling in each case.

The testing cooling times to reach thermal equilibrium were longer by afactor of 3-4 compared to the modeled cooling times as demonstrated inTable 4. This difference is small compared to difference in orders ofmagnitude relative to other processes such as SFL and lyophilization.The difference may be the result of uncertainty in the calibration ofthe temperature measurement, differences in definitions of the finaltemperature for the model and IR camera, and the release of the heat offusion of water which was not factored into the model. For extremelyrapid cooling rates of water, the water may form a glass with limitedcrystallization (67). As shown by data and calculation, a cooling rateof 10⁶ K/s is necessary to vitrify water (67-70). The 10² K/s coolingrate observed in TFF indicates that the latent heat of fusion may havebeen significant.

Nucleation and Growth Mechanisms versus Cooling Rate. To place the TFFresults in perspective, it is instructive to consider the boundaryconditions of extremely rapid vitrification/freezing in SFD and slowfreezing in lyophilization. Previously, the morphologies of lysozymepowders prepared by SFL and SFD were shown to be similar for dilute 5mg/mL lysozyme solutions. The SSAs were >100 m²/g for 50-100 nmspherical primary particles, despite a cooling rate of 10³ K/s for SFLversus 10⁶ K/s for SFD, as shown in Table 4 (36).

The freezing mechanism involves many simultaneous changes in theproperties of the unfrozen solution. As the water freezes, it changesconcentrations, pH, ionic strength, viscosity, diffusion coefficients,collisions between nucleated particles and geometric size and shape ofthe unfrozen solution. The growth rate of the protein particles dependsupon all of these factors, such that it would be challenging to developa model for the final particle size. The thin liquid channels betweenthe frozen water domains reduce the number of collisions between protein(sugar) particles and thus inhibit growth by coagulation, as shown inFIG. 8. Furthermore, the viscosity of the thin channels increasesrapidly to arrest particle growth and the channel fully freezes.Furthermore, the sugar in the water raises the viscosity over that ofpure water. For the case of slow cooling in lyophilization, the very lowdegree of supercooling creates relatively few nucleated ice domainscompared to the rapid cooling processes, leaving thick channels ofliquid solution between these domains. For a cooling rate of 1 K/min, asfor the case of slowly cooling a 5 mg/mL solution in a −20° C. freezer,the lyophilized particle sizes were on the order of 30-100 μm. In thesethick channels, the protein particles have sufficient time to aggregateand grow forming large particles before the channels are fully frozen.Although it is theoretically possible to mitigate this particle growthpartially by reducing the protein solution concentration significantlybelow 1 mg/mL, such low protein concentrations can lead to excessivelyophilization requirements (21).

In SFD, the present inventors found that exposure of the protein to thegas-liquid interface has a larger effect on protein stability than tothe ice-liquid or glassy water-liquid interface (19, 31, 32). It isunclear whether ice-liquid versus glassy water-liquid interfaces havedifferent effects on protein stabilities (19, 20, 71). As described byprevious references (68, 69), cooling rates on the order of 10⁶ K/s areneeded to vitrify water, but the cooling rate necessary forvitrification can be lowered in the presence of sugar in solution (67,70). For the slower cooling rates observed in TFF (10² K/s) relative toSFD, it is likely that ice particle domains instead of vitrified waterdomains are formed. The LDH activities were on the order of 100% forTFF. Thus, the present invention does not suggest that the ice-liquidinterface has a detrimental effect on protein stability.

For the 5 mg/mL lysozyme formulation at 223 K, the SSA was quite large,although modestly smaller than for SFD, and the particle sizes aftersonication were similar to those of both SFL and SFD as seen in Table 3.The lower cooling rate in TFF (10² K/s) compared to SFD (10⁶ K/s) andSFL (10³ K/s) was still sufficient to produce rapid nucleation and toprevent significant particle growth during freezing. However, for TFF,the size of the unfrozen channels was sufficiently thin and the increasein the viscosity of the unfrozen solution sufficiently fast to achievesimilar particle sizes and morphologies as for the moderately fasterprocess, SFL and much faster process, SFD. Thus, the extremely rapidlycooling rates in SFD were much faster than necessary to form submicronprotein particles. A similar conclusion was reached in the comparison ofSFL and SFD (32).

For 50 mg/mL highly concentrated solutions the larger volume fraction ofvitrified solute domains in the unfrozen water channels lead to agreater collision frequency and increased particle growth (36). Asobserved previously (36), the slower cooling rate in SFL compared to SFDleads to greater particle growth before the large unfrozen liquidchannels vitrify, leading to larger protein particles and lower powderSSAs (36). As shown in Table 3, the SSAs were similar for TFF and SFL.For these highly concentrated solutions, the larger particles formed inTFF (and SFL) versus SFD results from more time for growth in thethicker unfrozen channels. This limitation is typically not encounteredin rapid freezing processes, as most previous studies examined muchlower concentrations on the order of 5 mg/mL.

Minimization of gas-liquid interface in TFF process. The LDH stabilitieswere essentially 100% after TFF indicating that none of the steps,droplet falling, spreading and freezing, and drying caused a measurableloss in enzyme activity. From previous calculations (32) it was shownthat the exposure of the atomized droplets to the gas-liquid interfacewas an order of magnitude less in the SFL process (600 cm⁻¹) relative toSFD (6000 cm⁻¹) (19). This larger exposure to the gas-liquid interfaceresulted in lower LDH activities in SFD (32). In TFF the surfacearea/volume ratio of the gas-liquid interface of TFF (46 cm⁻¹) was 2orders of magnitude lower than in SFD, leading to far less proteinadsorption and aggregation. As shown in FIG. 9, the intermediate coolingrates in TFF and SFL offer a means to produce high surface areasubmicron particles as opposed to lyophilization, with smaller amountsof protein adsorption at gas-liquid interfaces compared to SFD resultingin higher protein stability.

Minimizing gas-liquid interface can improve protein stability bylimiting the amount of protein that can adsorb to the interface. Forsurface active radiolabeled proteins, the surface excess concentration,Γ, (72, 73) at full saturation for β-casein, lysozyme, and BSA were 2.6,3.0, and 3.3 mg/m², respectively (33, 72, 73). For LDH, we assumed asimilar value of approximately 3 mg/m2. For the top surface of a 12 mmdiameter film, where the surface area is 1.13×10-4 m², the totaladsorbed protein at equilibrium would be 3.4×10-4 mg. For a starting 3.6mm liquid droplet containing 0.25 mg/mL LDH, the total protein is6.2×10⁻³ mg. Therefore, if all of the protein reached the interface andwas denatured, the maximum decrease in protein activity would be 5.5%.The exposure of 1 s may not lead to full equilibrium adsorption.Furthermore, the increase in viscosity as a function of height and timewith freezing will arrest diffusion of protein to the air-waterinterface. For ˜10 μm diameter droplets in SFD, it was determined that25-30% of the total LDH in the droplet adsorbs to the gas-liquidinterface in only 0.4 ms (22). Denaturation of part of the adsorbedprotein is consistent with the significant decreases in protein activityobserved in the SFD process in Table 2.

The TFF process was utilized to produce 300 nm lysozyme particles withsurface areas on the order of 31-73 m²/g and 100% LDH activities.Despite a cooling rate of ˜10² K/s in TFF, the particle sizes andsurface areas were similar to those observed in the widely reportedprocess, spray freeze drying SFD, where cooling rates reach 10⁶ K/s. InTFF, the thin liquid channels between the ice domains were sufficientlythin and freezing rates of the thin channels sufficiently fast toachieve the similar particle morphologies. Therefore, the extremelyrapid cooling rate in the SFD process was not necessary to form thedesired submicron protein particles. Although LDH was exposed to thegas-liquid interface of the thin film for a maximum of ˜1 s in TFF, thesurface area/volume of 45 cm⁻¹ was sufficiently small that adsorptionproduced negligible aggregation and denaturation. Even if thisgas-liquid interface became saturated with protein, followed byirreversible denaturation, the maximum activity loss for a 0.25 mg/mLLDH formulation would be 5%. For SFD with a droplet size of 10 μm, themaximum loss could reach 25% in just 0.4 ms from diffusion to theinterface and adsorption (22), consistent with the significant decreasein enzyme activity (80%). In SFD, losses in protein stability have beenobserved in several previous studies (1, 11, 18, 19, 21). Although LDHstabilities are high in conventional lyophilization, cooling rates areon the order of 1 K/min resulting in large 30 to 100 μm sized particles(21). Thus, the intermediate cooling rate regime for TFF (and likewisefor SFL), relative to SFD and lyophilization, offers a promising routeto form stable submicron protein particles of interest in pulmonary andparenteral delivery applications.

Example 1

The solutions frozen using the TFF process has a final concentration of5 mg Lysozyme/mL solvent where the solvent was a water/ethanol mixtureat different concentration. The feed solution was then passed through a17 gauge needle at a flow rate of 4 mL/min falling from a height of 10cm onto a rotating stainless steel drum maintained at a temperature of223 K where the droplets were allowed to spread into disks and freeze.The frozen disks were then lyophilized using the standard lyophilizationprocedure described above. The resulting particle sizes (FIG. 11) weremeasured using the Malvern Mastersizer as described previously.

Example 2

Solutions in varying initial concentrations of lysozyme in water werefrozen as described above and then lyophilized to producemicroparticles. The frozen particles were frozen on the rotating drumand then scrapped off into small vial for individual dosages. Theparticle sizes produced were measured using the Malvern Mastersizer asdescribed above (FIG. 12).

FIGS. 13A to 13D compare the morphologies of TFF lysozyme prepared inglass vial versus TFF on a drum. Briefly, thin film freezing wasperformed directly in a vial that was cooled by submerging it partiallyin a liquid cryogenic fluid, or a fluid composed of dry ice and solvent.The feed was lysozyme at 5 mg/mL. The water in the frozen material wasthen removed by lyophilization of the vial. The product remained in thevial. A sample was removed from the vial and analyzed by scanningelectron microscopy. The morphology was similar to a sample prepareddirectly on a metal drum. The advantage of this technique is that TFFmay be performed directly in a glass vial to make particle withsubmicron features. The final dosage form may then be formulated byadding excipients to the particles in the vial.

TFF was performed using a feed of 150 mg/mL lysozyme in D.I. water. Oncethese samples were lyophilized, they were redispersed in acetonitrile tobreak the particles apart. The acetonitrile wa then removed by TFF. Thefinal particles after lyophilization were redispered in benzyl benzoateto form a stable suspension (FIG. 14).

The present invention demonstrates a simple, efficient and robustprocess for freezing either small (<1 mL) quantities of protein solutionor commercial quantities, that can produce stable submicron proteinparticles.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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1. A method for preparing micron-sized or submicron-sized particlescomprising: dissolving a water soluble effective ingredient in one ormore solvents; spraying or dripping solvent droplets such that theeffective ingredient is exposed to a vapor-liquid interface of less than500 cm⁻¹ area/volume; and contacting the droplet with a freezing surfacethat has a temperature differential of at least 30° C. between thedroplet and the surface, wherein the surface freezes the droplet into athin film with a thickness of less than 500 micrometers and a surfacearea to volume between 25 to 500 cm⁻¹.
 2. The method of claim 1, furthercomprising the step of removing the solvent from the frozen material toform particles.
 3. The method of claim 1, wherein the droplets freezeupon contact with the surface in about 50, 75, 100, 125, 150, 175, 200,250, 500, 1,000 and 2,000 milliseconds.
 4. The method of claim 1,wherein the droplets freeze upon contact with the surface in 50, 150, or500 milliseconds.
 5. The method of claim 1, wherein the droplet has adiameter between 0.1 and 5 mm at room temperature.
 6. The method ofclaim 1, wherein the droplet has a diameter between 2 and 4 mm at roomtemperature.
 7. The method of claim 1, wherein the droplet forms a thinfilm on the surface of between 50 and 500 micrometers in thickness. 8.The method of claim 1, wherein the droplets have a cooling rate ofbetween 50-250 K/s.
 9. The method of claim 2, wherein the particlesafter solvent removal have a surface area of 10, 15, 25, 50, 75, 100,125, 150 or 200 m²/gr.
 10. The method of claim 2, wherein the effectiveingredient is a protein or peptide and the particle has less than 50% ofthe peptide or peptide or protein at the particle surface.
 11. Themethod of claim 2, wherein the effective ingredient is a protein orpeptide and the particle has less than 25, 15, 10 or 5% of the peptideor peptide or protein at the surface.
 12. The method of claim 2, whereinthe particles are submicron in diameter.
 13. The method of claim 2,wherein the particles contain fibers less than one micron in diameter.14. The method of claim 1, wherein the surface is cooled by a cryogenicsolid, a cryogenic gas, a cryogenic liquid, a freezing fluid, a freezinggas, a freezing solid, a heat exchanger, or a heat transfer fluidcapable of reaching cryogenic temperatures or temperatures below thefreezing point of the solvent.
 15. The method of claim 1, wherein theeffective ingredient comprises an enzyme and the enzymatic activity ofthe enzyme is greater than 90%.
 16. The method of claim 1, wherein theeffective ingredient comprises a peptide or protein with an aggregationof the protein or peptide of less than 3%.
 17. The method of claim 1,wherein the temperature differential between the droplet and the surfaceis at least 50° C.
 18. The method of claim 1, further comprising furthercomprising at least one of excipients.
 19. A pharmaceutical formulationcomprising drug particles prepared by the method of claim
 1. 20. Amethod for preparing micron-sized or submicron-sized solvent particlescomprising: spraying or dripping droplets of a water soluble peptide orprotein in a solvent, wherein the droplet is exposed to an vapor-liquidinterface of less than 250 cm⁻¹ area/volume; contacting the droplet witha freezing surface that has a temperature differential of at least 30°C. between the droplet and the surface, wherein the droplet freezes intoa thin film with a thickness of less than 500 micrometers and a surfacearea to volume between 25 to 500 cm⁻¹.
 21. The method of claim 20,further comprising the step of removing the solvent from the frozenmaterial to form particles.
 22. The method of claim 20, the solventfurther comprises at least one of sugars, phospholipids, surfactants,polymeric surfactants, vesicles, polymers, including copolymers andhomopolymers and biopolymers, dispersion aids, and serum albumin. 23.The method of claim 20, wherein the peptide or protein comprises anenzyme and the enzymatic activity of the enzyme is greater than 90%. 24.The method of claim 20, wherein the peptide or protein aggregation isless than 3%.
 25. The method of claim 20, wherein the temperaturedifferential between the solvent and the surface is at least 50° C. 26.The method of claim 20, wherein the particle has less than 50% of thepeptide or protein at the surface.
 27. The method of claim 20, whereinthe particle has less than 25, 15, 10 or 5% of the peptide or protein atthe surface.
 28. A pharmaceutical formulation comprising drug particlesprepared by the method of claim
 20. 29. A method for preparingmicron-sized or submicron-sized particles comprising: preparing anemulsion comprising a water soluble effective ingredient in solution;spraying or dripping droplets of the solution such that the effectiveingredient is exposed to an vapor-liquid interface of less than 50 cm⁻¹area/volume; and contacting the droplet with a freezing surface that hasa temperature differential of at least 30° C. between the droplet andthe surface, wherein the surface freezes the droplet into a thin filmwith a thickness of less than 500 micrometers and a surface area tovolume between 25 to 500 cm⁻¹.
 30. A system for preparing solventparticles comprising: a solvent source composed of one or more solvents;a vessel containing a cryogenic liquid selected from cryogenic liquidselected from the group consisting of carbon dioxide, nitrogen, ethane,propane, helium, argon, or isopentane; and an insulating nozzle havingan end and a tip, wherein the end of the nozzle is connected to thesolvent source and the tip is placed at or below the level of thecryogenic liquid.
 31. The system of claim 30, wherein the solutionsource further comprises water, at least one organic solvent, or acombination thereof.
 32. The system of claim 30, wherein the organicsolvent is elected from the group consisting of ethanol, methanol,tetrahydrofuran, acetonitril acetone, tert-butyl alcohol, dimethylsulfoxide, N,N-dimethyl formamide, diethyl ether, methylene chloride,ethyl acetate, isopropyl acetate, butyl acetate, propyl acetate,toluene, hexanes, heptane, pentane, and combinations thereof.
 33. Amethod for freezing comprising: spraying one or more solvents through aninsulating nozzle located above, at or below the level of a cryogenicmaterial, wherein the solvent particles have a size range of 10 nm to 10microns.
 34. The solvent particle of claim 33, wherein the surface areaof the particles is greater than 50 m²/g.
 35. The solvent particle ofclaim 33, wherein the cryogenic material comprises a liquid, a gas, asolid or a surface.
 36. The solvent particle of claim 33, wherein theone or more solvents comprises a first solvent that is less volatilethan a second solvent, wherein the more volatile solvent is removed butnot the second solvent.
 37. The solvent particle of claim 33, whereinthe one or more solvents comprises a first solvent that is less volatilethan a second solvent, wherein the more volatile solvent is removed byevaporation, lyophilization, vacuum, heat or chemically.
 38. A solventparticle produced by method of claim 33 wherein the size of theparticles is less than 10 microns.
 39. A single-step, single-vial methodfor preparing micron-sized or submicron-sized particles comprising:reducing the temperature of a vial wherein the vial has a temperaturedifferential of at least 30° C. between the solvent and the vial; andspraying or dripping solvent droplets of a water soluble effectiveingredient dissolved in one or more solvents directly into the vial suchthat the effective ingredient is exposed to a vapor-liquid interface ofless than 500 cm⁻¹ area/volume, wherein the surface freezes the dropletinto a thin film with a thickness of less than 500 micrometers and asurface area to volume between 25 to 500 cm⁻¹.
 40. The method of claim39, wherein the droplets freeze upon contact with the surface in about50, 75, 100, 125, 150, 175, 200, 250, 500, 1,000 and 2,000 milliseconds.41. The method of claim 39, wherein the droplets freeze upon contactwith the surface in about 50 to 500 milliseconds.
 42. The method ofclaim 39, wherein the droplets freeze upon contact with the surface inabout 50 to 150 milliseconds.
 43. The method of claim 39, wherein thedroplet has a diameter between 0.1 and 5 mm at room temperature.
 44. Themethod of claim 39, wherein the droplet has a diameter between 2 and 4mm at room temperature.
 45. The method of claim 39, wherein the dropletforms a thin film on the surface of between 50 and 500 micrometers inthickness.
 46. The method of claim 39, wherein the droplets have acooling rate of between 50-250 K/s.
 47. The method of claim 39, whereinthe vial is cooled by a cryogenic solid, a cryogenic gas, a cryogenicliquid, a freezing fluid, a freezing gas, a freezing solid, a heatexchanger, or a heat transfer fluid capable of reaching cryogenictemperatures or temperatures below the freezing point of the solvent.48. The method of claim 39, wherein the vial, the water solubleeffective ingredient and the one or more solvents are pre-sterilizedprior to spraying or dripping.
 49. The method of claim 39, wherein thestep of spraying or dripping is repeated to overlay one or more thinfilms on top of each other to fill the vial to any desired level up tototally full.
 50. The method of claim 39, further comprising the step ofresuspending the composition in a solvent in an individually dosed vialto create a suspension or solution for delivery of the effectiveingredient.